Structure and function of the protein chaperone Tti2

蛋白伴侣 Tti2 的结构和功能

• 批准号: RGPIN-2015-04394
• 负责人: Brandl, Christopher
• 金额: $ 2.48万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=612939
• 关键词: Structure; function; protein; chaperone; Tti2

The folding of many proteins requires chaperones. Chaperones also untangle misfolded proteins or target them for degradation. Chaperones are particularly critical in the response to stress; thus global warming is making mechanisms to modulate their activity genetically or chemically increasingly valuable. Our studies focus on the TTT chaperone complex. The components of this complex, Tel2, Tti1 and Tti2, are essential in eukaryotes. Using genetic selections in S. cerevisiae we identified mutations in TTI2 that enhance the folding of a member of the PIKK (Phosphatidylinositol 3-kinase-related kinase) protein family. This effect results from partial loss of Tti2 function, leading us to hypothesize that Tti2 targets slowly folding proteins for degradation. Similar mutations are not found in TEL2 or TTI1, suggesting that Tti2's function may be unique. The goal of our research program is to determine the function of the components of the TTT-complex. An initial emphasis will be placed on Tti2, because of its potentially unique activities and since we have many of the key tools for its analysis. Our studies use yeast because the combination of experimental approaches yeast allow is ideal for solving fundamental biological problems. Specific objectives for this five-year period are: 1. Identify the protein interaction networks of the components of the TTT-complex. The functions of Tel2, Tti1 and Tti2 will be more clearly defined when their interacting partners are determined. We will determine the interaction partners of the three proteins using biochemical approaches. The significance of novel interactions will be examined by mapping the interaction surface through mutagenesis, then introducing the mutagenized alleles into cells. 2. Characterize the structure/function relationships of Tti2. Using unigenic evolution we have identified clusters of invariant residues in Tti2. These residues will be mutated and their phenotypic consequences determined by plasmid shuffling in a tti2 knockout strain we constructed. The effects of the mutations on Tti2 expression, localization, folding of the PIKK proteins, and protein-protein interactions will be examined. Alleles that reduce cell growth will be valuable tools for suppressor genetic studies that will guide experiments to determine the function of Tti2. 3. Identify the “client” proteins for Tti2. The importance of the TTT proteins in the folding of members of the PIKK family has been demonstrated. Whether other clients exist is unknown. We will search for proteins whose expression depends on Tti2 and Tel2 using conditional alleles and protein identification through stable isotope labeling with amino acids in cell culture. A second approach to identify both targets and interacting proteins will be through the BioID strategy. This method was designed for mammalian cells, so we will start by identifying targets of mammalian Tti2 in cell culture.

许多蛋白质的折叠需要伴侣。伴侣也是脱包的蛋白质或靶向降解的蛋白质。伴侣在应激的反应中尤其重要。因此，全球变暖正在制定机制，以调节其一般或化学越来越有价值的活动。我们的研究集中于TTT伴侣复合体。这种复合物，Tel2，TTI1和TTI2的组成部分在真核生物中至关重要。使用酿酒酵母中的遗传选择，我们鉴定了TTI2中的突变，从而增强了Pikk（磷脂酰肌醇3-激酶相关激酶）蛋白家族的折叠。这种影响是由于TTI2功能的部分丧失而导致的，这使我们假设TTI2靶向缓慢折叠蛋白以降解。在Tel2或TTI1中未发现类似的突变，这表明TTI2的功能可能是唯一的。我们的研究计划的目的是确定TTT复合物组件的功能。由于其潜在的独特活动，并且由于我们拥有许多关键工具进行分析，因此最初的重点将放在TTI2上。我们的研究之所以使用酵母，是因为酵母允许的实验方法的组合是解决基本生物学问题的理想选择。这个五年的特定对象是： 1。识别TTT复合物组件的蛋白质相互作用网络。当确定其相互作用伙伴时，Tel2，TTI1和TTI2的功能将更清楚地定义。我们将使用生化方法确定三种蛋白质的相互作用伙伴。新相互作用的意义将通过通过诱变将相互作用表面映射，然后将诱变等位基因引入细胞来检查。 2。表征TTI2的结构/功能关系。使用单基因进化，我们确定了TTI2中不变保留簇。这些保留将被突变，它们的表型后果是由我们构建的TTI2敲除菌株中的质粒改组确定的。将研究突变对TTI2表达，定位，Pikk蛋白的折叠和蛋白质 - 蛋白质相互作用的影响。减少细胞生长的等位基因将是抑制遗传研究的有价值的工具，可以指导实验确定TTI2的功能。 3。确定TTI2的“客户端”蛋白。已经证明了TTT蛋白在Pikk家族成员折叠中的重要性。是否存在其他客户是未知的。我们将使用条件等位基因和蛋白质鉴定来搜索其表达取决于TTI2和TEL2的蛋白质，该蛋白质通过稳定的同位素标记在细胞培养中用氨基酸标记。识别靶标和相互作用蛋白质的第二种方法将是通过生物分子策略。该方法是为哺乳动物细胞设计的，因此我们将首先识别细胞培养中哺乳动物TTI2的靶标。